1. Field of the Invention
The present invention relates to electrodes for detecting nucleic acid hybridization, and to methods of making and using such electrodes.
2. Description of the Related Art
Recent advances in surface modification techniques have facilitated many new methods for bioassay technology, particularly when coupled with sophisticated fluorescent detection technologies. For example, gene expression analysis (Schena, M. et al., Science 1995, 270, 467); sequencing of genomic DNA on high-density arrays (Chee, M. et al., Science, 1996, 274, 610); and the detection of nucleic acids to identify infectious organisms (Spargo, C. A. et al., Molecular and Cellular Probes 1993, 7, 395; Martin, W. J. in The Polymerase Chain Reaction; Mullis, K. B.; Ferre, F.; Gibbs, R. A, eds.; 1994, 406-417. Berkhauser, Boston) have the potential for superior selectivity and sensitivity when compared to preexisting culture or immunoassay-based methods. The disclosures of each of the patents and publications referred to herein are incorporated herein by reference. While these systems present significant advancements, they still involve extensive pretreatment steps and the use of expensive fluorescent microscopes.
The electrochemical detection of nucleic acids provides an alternative to fluorescent bioassay techniques that potentially eliminates the need for labeling (Johnston, D. H. et al., Metal Ions Biol. Syst. 1996, 33, 297; Johnston, D. H.; Cheng, C. C. et al., Inorg Chem. 1994, 33, 6388; Steenken, S. et al., J. Am. Chem. Soc. 1997, 119, 617; Johnston, D. H. et al., J. Am. Chem. Soc. 1995, 117, 8933; and Johnston, D. H. et al., J. Phys. Chem. 1996, 100, 13837).
The invention herein utilizes the discovery that the guanine nucleobases of polymeric DNA produce an array of redox-active labels suitable for ultrasensitive detection that, in conjunction with ultramicroelectrode methods, provide a method for detecting many physiologically relevant nucleic acids prior to PCR amplification. Incorporation of individual microelectrodes into an array allows production of low-cost, rapid-throughput devices with high-density, multiplexed sensor arrays.
Nucleic acids can be detected in solution via catalytic oxidation of guanine bases using Ru(bpy).sub.3.sup.2+ as the mediator (Johnston, D. H. et al., Metal Ions Biol. Syst. 1996, 33, 297; Johnston, D. H. et al., Inorg. Chem. 1994, 33, 6388; Johnston, D. H. et al., J. Am. Chem. Soc. 1995, 117, 8933; and Johnston, D. H. et al., J. Phys. Chem. 1996, 100, 13837). In solution, Ru(bpy).sub.3.sup.2+ exhibits a reversible redox couple at 1.05 V similar to the oxidation potential observed for guanine. Addition of guanine-containing DNA to a solution of Ru(bpy).sub.3.sup.2+ leads to catalytic enhancement in the oxidation current according to a two-step mechanism: EQU Ru(bpy).sub.3.sup.2+.fwdarw.Ru(bpy).sub.3.sup.3+ +e- EQU Ru(bpy).sub.3.sup.3+ +DNA.fwdarw.DNA.sub.ox +Ru(bpy).sub.3.sup.2+
where DNA.sub.ox represents a DNA molecule where guanine has undergone a one electron oxidation.
As set forth in the co-pending parent application (Ser. No. 08/667,338), hybridized DNA may be immobilized on a solid support, and the oxidizing agent then reacted with the hybridized DNA by immobilizing the oxidizing agent on the same solid support and immersing the solid support in a solution under conditions sufficient to permit the oxidation-reduction reaction of the oxidizing agent and a preselected base to occur.
As set forth in co-pending application Ser. No. 08/950,503 (polymer-electrode application), additional schemes in which DNA immobilized onto poly(ethylene terepthalate) or PET membranes and DNA probes attached directly to Indium-Tin Oxide (ITO) electrodes were used for detection of complementary DNA with application as a PCR amplicon bioassay (Napier, M. E. et al., Langmuir 1997, 13, 6342; Napier, M. E. et al., H. H. Bioconjugate Chem. 1997, 8, 906). In essence, the body of previous investigation has focused on two scenarios: 1) solution mediators with solution DNA and 2) solution mediators with immobilized DNA.
Electrochemical copolymerization has been used to prepare matrices. For example, a DNA matrix has been prepared on an electrode surface via an electrochemically directed copolymerization of pyrrole and oligonucleotides bearing a pyrrole group, together with radiolabeling of the oligonucleotides to detect hybridization (Livache, T. et al., Nucleic Acids Res. 1994, 22, 2915; Roget, A. et al., Nucleosides & Nucleotides 1995, 14, 943). Also, biosensors have been designed using an electroactive polypyrrole functionalized with an oligonucleotide probe (Korri-Youssoufi, H. et al., J. Am. Chem. Soc., 1997, 119, 7388).
While Livache and Roget use a radioactive label to detect the DNA attached to the electropolymerized film and Korri-Yousoufi monitors the potential of the film itself to indirectly detect DNA hybridization, the invention herein allows for the direct detection of DNA attached to the electropolymerized film via faradaic current from guanine. Thus, the Korri-Yousoufi approach is a potentiometric method, whereas the present method is amperometric. The polymers described herein provide a catalyst for the transfer of electrons from the nucleic acid to the electrode that enables detection of faradaic current, which would be too slow to provide a practical signal without the immobilized mediator. In addition the films of the invention are electrochemically inert in the region 0-0.9 V, whereas the prior polypyrrole films are reactive in this region.
The patent of Yachynych (U.S. Pat. No. 5,540,828) provides a method for making electrodes for protein recognition by electropolymerizing oxidatively. In contrast to Yachynych, the invention herein is used for nucleic acids, in the invention herein polymers are formed reductively rather than oxidatively during polymerization, the invention herein utilizes vinyl-containing polymers, and the metal complex used is both an initiator for electropolymerization and immobilized mediator.
It is therefore an object of the invention to provide surface-modified electrodes for the detection of electron transfer events at potentials near those observed for guanine or other preselected bases in DNA or RNA.
It is a further object of the invention to immobilize a mediator, namely the Ru.sup.2+ center, for use in the electrochemical detection of guanine bases present in both solution and surface immobilized species.
It is a further object of the invention to use electropolymerization to generate electrodes modified with probes that give greater oxidative current upon hybridization to targets containing larger numbers of preselected bases.
It is a further object of the invention to provide an agent that acts both as initiator for electropolymerization and as a mediator for oxidation of a preselected base.
Other objects and advantages will be more fully apparent from the following disclosure and appended claims.